RESEARCH ARTICLE
Gai analysis: An e ec i e ool o mechanically
moni o he bone egene a ion o c i ical-
sized de ec s in issue enginee ing
applica ions
Pablo Bla
´zquez-Ca monaID
1,2
*, Juan Mo a-Macı
´as
2,3
, Juan Mo gazID
4
, Ma ı
´a del
Ma G anados
4
, Jaime Domı
´nguez
1,2
, Es he Reina-Romo
1,2
1Depa men o Mechanical and Manu ac u ing Enginee ing, Escuela Te
´cnica Supe io de Ingenie ı
´a,
Uni e sidad de Se illa, Se ille, Spain, 2Ins i u o de Biomedicina de Se illa (IBiS), Uni e si y o Se ille,
Se ille, Spain, 3Depa men o Mining, Mechanical, Ene gy and Building Enginee ing, Escuela Te
´cnica
Supe io de Ingenie ı
´a, Uni e si y o Huel a, Huel a, Spain, 4Depa men o Animal Medicine and Su ge y,
Uni e sidad de Co
´ doba, Campus Uni e si a io de Rabanales, Co
´ doba, Spain
*pbca [email protected]
Abs ac
In oduc ion
Tissue enginee ing has eme ged as an inno a i e app oach o ea c i ical-size bone
de ec s using biocompa ible sca olds, hus a oiding complex dis ac ion su ge ies o limi ed
s ock g a s. Con inuous egene a ion moni o ing is essen ial in c i ical-size cases due o he
equen appea ance o non-unions. This wo k e alua es he po en ial clinical use o gai
analysis o he mechanical assessmen o a issue enginee ing egene a ion as an al e na-
i e o he adi ional and ha dly conclusi e manual o adiological ollow-up.
Ma e ials and me hods
The 15-mm me a a sal agmen o eigh emale me ino sheep was su gically eplaced by a
bioce amic sca old s abilized wi h an ex e nal ixa o . Gai es s we e pe o med weekly by
making he sheep walk on an ins umen ed gangway. The e olu ion o di e en kinema ic
and dynamic pa ame e s was analyzed o all he animal’s limbs, as well as asymme ies
be ween limbs. Finally, po en ial co ela ion in he eco e y o he gai pa ame e s was e al-
ua ed h ough he linea eg ession models.
Resul s
A e su ge y, he ope a ed limb has an al e ed way o ca ying body weigh while walking.
I s loading capaci y was signi ican ly educed as he s ance phases we e sho e and less
impulsi e. The non-ope a ed limbs compensa ed o his mobili y de ici . All pa ame e s
we e no malizing du ing he consolida ion phase while he bone callus was simul aneously
mine alizing. The esul s also showed high le els o asymme y be ween he ope a ed limb
and i s con ala e al, which exceeded 150% when analyzing he impulse a e su ge y. Gai
eco e y signi ican ly co ela ed be ween symme ical limbs.
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PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0296510 Decembe 29, 2023 1 / 18
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OPEN ACCESS
Ci a ion: Bla
´zquez-Ca mona P, Mo a-Macı
´as J,
Mo gaz J, G anados MdM, Domı
´nguez J, Reina-
Romo E (2023) Gai analysis: An e ec i e ool o
mechanically moni o he bone egene a ion o
c i ical-sized de ec s in issue enginee ing
applica ions. PLoS ONE 18(12): e0296510. h ps://
doi.o g/10.1371/jou nal.pone.0296510
Edi o : Yaodong Gu, Ningbo Uni e si y, CHINA
Recei ed: Sep embe 24, 2023
Accep ed: Decembe 13, 2023
Published: Decembe 29, 2023
Pee Re iew His o y: PLOS ecognizes he
bene i s o anspa ency in he pee e iew
p ocess; he e o e, we enable he publica ion o
all o he con en o pee e iew and au ho
esponses alongside inal, published a icles. The
edi o ial his o y o his a icle is a ailable he e:
h ps://doi.o g/10.1371/jou nal.pone.0296510
Copy igh : ©2023 Bla
´zquez-Ca mona e al. This is
an open access a icle dis ibu ed unde he e ms
o he C ea i e Commons A ibu ion License,
which pe mi s un es ic ed use, dis ibu ion, and
ep oduc ion in any medium, p o ided he o iginal
au ho and sou ce a e c edi ed.
Da a A ailabili y S a emen : All ele an da a a e
wi hin he pape and i s Suppo ing In o ma ion
iles.
Conclusions
Gai analysis was p esen ed as an e ec i e, low-cos ool capable o mechanically p edic -
ing he egene a ion o c i ical-size de ec s ea ed by issue enginee ing, as compa ing
egene a ion p ocesses o no el sca olds. Despi e he p og essi e no maliza ion as he cal-
lus mine alized, he bea ing capaci y educ ion and he asymme y o he ope a ed limb
we e mo e signi ican han in o he o hopedic al e na i es.
In oduc ion
Bone is a highly dynamic hie a chical composi e wi h homeos a ic mechanisms in cha ge o
i s con inuous emodeling and egene a ion in he e en o de ec s caused by umo s, aumas,
o in ec ions. Ne e heless, he egene a i e capaci y o ha d issue is no unlimi ed. These
de ec s ha e a size uppe limi om which his igid o gan does no heal spon aneously, usually
leading o non-unions [1]. Thus, he ea men o c i ical-sized bone de ec s is s ill oday one
o he signi ican challenges o acu e ca e su geons. To add ess his issue, hey mus eso o
o he clinical s a egies, including hose based on he Iliza o me hod (dis ac ion os eogene-
sis) o he inco po a ion o subs i u es. Fo ins ance, bone anspo us s on he long-s and-
ing dis ac ion os eogenesis o g adually mo e an os eo omized su ounded bony agmen
owa ds he posi ion o he o iginal de ec while simul aneously o ming a bone callus on he
o he side, he docking si e [2–4]. Howe e , his egene a ion p ocess ca ies inhe en isks,
pa icula ly conce ning iscoelas ic and s uc u al al e a ions in he neighbo ing so issues
(e.g., skin, endons, o muscles) [5–8]. Ano he adi ional solu ion lies in inco po a ing au o-
genic and allogenic bone g a s o encou age cell egene a i e ac i i y in he gap, mainly ascu-
la ized ee ibula o Papineau open cancellous bone g a ing [9–11]. Despi e being he mos
sui able subs i u e conce ning biocompa ibili y, os eoinduc i e and os eoconduc i e p ope -
ies, he a ailabili y o ha es ed au og a ing issue is limi ed, and he allog a ’s g owing clini-
cal demand exceeds he dono s ock [12,13]. Since no long ago, esea ch o no el al e na i es
o issue ans e has been ongoing h ough issue enginee ing (TE) [14]. TE is ocused on
inding he s anda d subs i u e ma e ial and s uc u e o e ec i ely emula e he in ica e
mic oen i onmen o na i e bone issue and u he p omo e egene a ion [15,16]. In his
ield, addi i e manu ac u ing echniques ha e gained popula i y o hei e sa ili y in building
s able h ee-dimensional po ous sca olds wi h a b oad ange o in e nal mic oa chi ec u es o
in es iga e nume ically. This lexibili y ma e ialized in many scien i ic wo ks cu en ly ocus
on he op imiza ion o he mic os uc u e o sca olds wi h di e en ypes o ma e ials beyond
he adi ional biomedical me als, including bioce amics o polyme s [16–18]. Al hough sca -
olds ha e been implan ed wi h easonable success in he epai o small bone de ec s [19,20],
hei success wi h c i ical-sized ones emains limi ed [21,22].
A e an o hopedic su ge y o a c i ical-sized bone de ec , con inuous moni o ing o he
bone egene a ion p ocess is ad isable, especially in he ea ly weeks when bone in ec ions o
non-unions equen ly appea . In his line, he p og ess o a bone healing p ocess is ypically
moni o ed by plain ilm adiology and densi ome ic me hods [23–25]. Beyond being accom-
panied by equen exposu e o adia ion, he lack o con inui y in his ollow-up cons an ly
esul s in quali a i e conclusions and ime lags in c i ical decision-making, including he need
o ein e en ion o ixa ion emo al. These delays occu since de ec mine aliza ion is p i-
ma ily e lec ed in adiological images ime a e he eal inc ease in bone callus s i ness
[26,27]. Poo co ela ions o adiog aphic measu emen s wi h he issue mechanical p ope ies
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Funding: This p ojec was suppo ed by he
Minis e io de Ciencia e Inno acio
´n (Go e nmen o
Spain) h ough he g an numbe PID2020-
113790RB-I00 awa ded o JMM and ERR, h ps://
www.aei.gob.es/con oca o ias/buscado -
con oca o ias/p oyec os-idi-2020-modalidades-
e os-in es igacion-gene acion, and by he Jun a
de Andalucı
´a (FEDER-UHU, P og ama Ope a i o
FEDER de Andalucı
´a 2014-2020) h ough he g an
numbe UHU-202058 awa ded o JMM, h ps://
www.agenciaandaluzadelaene gia.es/es/
inanciacion/incen i os-2017-2020/ ondo- ede -
2014-2020. The unde s had no ole in s udy
design, da a collec ion and analysis, decision o
publish, o p epa a ion o he manusc ip " was
included in he co e le e o he e iewed
manusc ip .
Compe ing in e es s: The au ho s ha e decla ed
ha no compe ing in e es s exis .
ha e been consequen ly epo ed in he li e a u e [27]. Thus, despi e he de elopmen o sco -
ing sys ems and indexes o e alua e and compa e se s o adiog aphs [23,24], in p ac ice, clini-
cians end bo h o o e es ima e o unde es ima e a lowe callus s eng h han i eally is [28].
O he adi ional in i o manual me hods, such as clinical symp oms ( ende ness o pain
while bea ing weigh ) o he mobili y e iew o he ea ed bone, us he clinician’s abili y o
in e p e examina ions and do no ensu e he p ope ossi ica ion in any case [29]. In he issue
enginee ing ield, he opaci y o i s base ma e ial, i s small po e sizes, and he gene ally high
appa en s i ness o he s uc u e could hinde pe o ming a ealis ic analysis o he naï e is-
sue o ma ion inside om adiological o manual assessmen s, especially in hose c i ical i s
pos -ope a i e weeks. In ecen decades, enginee s ha e been de eloping al e na i es ha o e
quan i a i e mechanical and s uc u al pa ame e s o p edic bone egene a ion indi ec ly.
Loads h ough he ixa ion, in e agmen a y displacemen s, s ains, o acous ic emissions col-
lec ed by means o a wide a ie y o senso s allow es ima ing he bone callus s i ness in i o
and mechanically compa ing su gical p o ocols and bone egene a ion app oaches [5,30–35].
Howe e , mos ins umen ed sys ems can only be coupled in o ex e nal ixa o s and equi e
complex and expensi e acquisi ion equipmen no widely a ailable oday in he clinical ou-
ine. In ib a ional measu emen s, he ea ed bone is also equi ed o be subcu aneous
[23,32].
As ano he op ion, gai analysis has been used o ollow up on kinema ics [36], elec omyo-
g aphic (EGM) [37], o load-bea ing pa ame e s [38–40] po en ially modi ied by o hopedic
su ge y. Du ing kinema ics es s and EGM, measu emen s o e comple e in o ma ion ega d-
ing he posi ion, o ien a ion, and elec ical ac i i y o many body segmen s a he expense o
he ime-consuming asks o a aching skin ma ke s o elec odes o he pa ien s’ bodies
[36,37]. Among all he po en ially collec ible biomechanical pa ame e s, some au ho s ha e
de eloped algo i hms o op imize he bes se ha iden i ies signi ican di e ences in gai pa -
e ns unde he speci ic condi ions o each s udy [41,42]. By cons, he g ound eac ion o ce
(GRF) measu emen allows analyzing o ce- ime cu es collec ed by load-bea ing pla o ms
du ing he pa ien ’s s ance phases. The simplici y and he possibili y o di ec ly examining
hese da a in i o wi h no complex da a pos -p ocessing make i he mos ex apola ed- o-
clinic me hod o s udy he dynamic e olu ion o he lowe limbs a e o hopedic ea men s
o su ge ies. GRF has also been epo ed o e lec he eco e y o appa en s i ness a he
de ec si e un il he ull unc ional eco e y in ac u e healing and dis ac ion os eogenesis
p ocesses [38–40]. I also makes i possible o compa e he impac o su ge y on bea ing capac-
i y be ween o hopedic ea men s, as well as he speed o eco e y in each case. In addi ion,
clinical decisions based on GRF da a a e p o en mo e eliable han hose aken om kinema ic
o EGM pa ame e s [43,44]. As a as he au ho s a e conce ned, gai analysis has no been
es ed in bone issue enginee ing applica ions on lowe limbs, and i s use ulness in assessing
he p ope egene a ion o bony de ec s ea ed wi h sca olds emains unknown.
In his line, his wo k aims o e i y he use ulness o gai analysis in i o o assessing he
bone egene a ion o weigh -bea ing c i ical-sized de ec s ea ed wi h bioce amic sca olds
ex e nally s abilized. The p oposed moni o ing me hodology is clinicians- iendly h ough
low-cos de ices whose collec ed da a do no equi e complex pos -p ocessing asks. This wo k
also del ed in o he po en ial co ela ion be ween he di e en analyzed gai pa ame e s in he
ope a ed and non-ope a ed limbs h oughou he TE mine aliza ion phase as a means o sim-
pli y gai analysis in eal clinical scena ios. Besides, he eco e y mode o he s udied pa ame-
e s, including da a om he con ala e al limb, will be compa ed wi h o he equi alen
o hopedic ea men s on he same bone model (e.g., bone anspo ) o in es iga e he unc-
ional ad an ages o each egene a ion p ocess.
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Ma e ials and me hods
Animals and bone model
TE expe imen s we e pe o med in i o on eigh igh -back o ine me a a si o adul emale
Me ino sheep (n = 8). A con ol g oup o 3 non-ope a ed animals, andomly selec ed, we e
included in he s udy o con as esul s wi h he expe imen al g oup di ec ly (n = 3). The e-
o e, expe imen s we e ca ied ou in a o al o 11 animals. Sample size was selec ed acco ding
o he s anda d de ia ion and s a is ical es s ound in a p e ious s udy [39]. This s udy and i s
p o ocol we e p e iously app o ed by he Animal E hics o he Uni e si y o Co
´ doba (P o o-
col Numbe : 2021PI/21) and gua an eed du ing su gical in e en ions and expe imen al
phases he animal’s wel a e in s ic acco dance wi h he ARRIVE guidelines, he Eu opean
(2010/63/UE) and na ional (RD 1201/2005) egula ions on animal expe imen a ion, hus
a oiding s ess ul si ua ions. A comple ed copy o he Full ARRIVE 2.0 Guidelines checklis is
p o ided in he S1 Checklis o he Suppo ing In o ma ion. The selec ed mammalian model
has nume ous ad an ages. Sheep a e docile, low-p iced, and ha e a bone composi ion and
body weigh compa able o humans [45–47]. These simila i ies allow o easie ex apola ion
o conclusions o eal clinical cases. The me a a sus was chosen as bone mode due o i s hin
laye o su ounding so issues, inding exclusi ely pe ios eal issue on he la e al and medial
sides. This ana omical ad an age allowed a sa e and mo e accessible issue enginee ing su -
ge y. The animals we e ob ained om a a m o esea ch and we e ma ked on he wool o
a oid con ounde s. They we e heal hy, wi h an adequa e accina ion and dewo ming p o ocol.
Sheep wi h an adequa e me a a sal leng h we e selec ed o he s udy (>12 cm). They we e s a-
bled 15 days be o e he s a in he esea ch cen e acili ies o an adap a ion pe iod whe e all
expe imen s we e ca ied ou . Thei acili ies we e adap ed o p o ide o hei needs, includ-
ing shade, com o able lying a eas, and secu e encing o keep hem uns essed and sa e. In he
ope a ed g oup, a 15-mm c i ical-size bone segmen o igh me a a sus was su gically eplaced
by a subjec -speci ic bioce amic sca old. Fo compa a i e easons, his bone de ec size was
selec ed based on he size in o he o ine bone egene a ion expe imen s in he li e a u e
[4,5,39]. The de ec was ex e nally s abilized by a modula Iliza o - ype ex e nal ixa o . This
ex e nal solu ion minimizes body in asion compa ed o in e nal pla es and spli s. Hence, hey
educe he so issue damage, leading o an ea ly eco e y o he animals’ mobili y and a lowe
impac on he analyzed gai pa ame e s in he weeks immedia ely ollowing he in e en ion.
When he expe imen al pa o he s udy concluded, he animals we e eu hanized by an o e -
dose o sodium pen oba bi al IV Eu hasol1. The ollowing subsec ions co e he design and
manu ac u ing p ocess o he sca old, he s eps o he o hopedic su ge y, de ails abou he in
i o gai expe imen s, and he adiological assessmen . In he “Su ge y p ocedu e” subsec ion,
all ele an s eps aken o amelio a e animal su e ing du ing he esea ch a e also de ailed.
Design and ab ica ion o he sca old
The sca old was designed wi h he subjec -speci ic geome y o he bone agmen o be
eplaced. Fo his, compu ed omog aphy was pe o med on he igh hindlimb o he sheep
be o e su ge y ( oxel size 0.12 x 0.12 x 0.60 mm). A mul iplana ha d issue h esholding was
applied o each me a a sal scan using he so wa e InVesalius1(Rena o A che In o ma ion
Technology Cen e , Ama ais, B azil), which enabled o gene a e a 3D bone econs uc ion
om he image s ack, as shown in Fig 1A. A e wa d, an in e media e 13-mm bone segmen
was sliced om he me a a sal geome y employing he solid modeling CAD so wa e Space-
Claim1(SpaceClaim Co po a ion, Conco d, MA, USA), and i s inne medulla y ca i y was
illed. Two building modi ica ions we e also ca ied ou on his ini ial geome y o imp o e
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he sca old s abili y in he de ec and speed up he egene a i e esponse (see Fig 1A). Fi s ly,
a coupling cylinde (4 mm Ø; 2 mm leng h) was added o e one end ace o immobilize he
s uc u e in i o in he dis al bone ma ow. This couple would p e en he sca old om
o a ing o mo ing du ing he animals’ daily ac i i y, which could in e e e wi h he p ope
g ow h o he naï e issue. A he o he ex eme, he solid was hollowed ou by ano he cylin-
de (4 mm Ø; 10 mm leng h) o g a ing, as u he explained. A obocas ing de ice (3-D Inks
S ill-wa e 1, Tulsa, Oklahoma, USA), an ex usion-based 3D- p in ing echnique, was
selec ed. I wo ked by deposi ing a 45 ol% hyd oxyapa i e slu y o ming a ce amic ne wo k
o pe pendicula ly o ien ed laye s o ba s, as illus a ed in Fig 1B. This ma e ial was chosen
since bioce amics a e p o en o enhance naï e issue g ow h by egula ing os eoblas p oli e a-
ion and di e en ia ion while he s uc u e is eabso bed [48]. F om p e ious wo ks in he li -
e a u e, he concen a ion o colloidal suspensions in he 3D-p in ing ink (45 ol%) was
op imized o ob ain he sui able iscoelas ic p ope ies o an e ec i e deposi ion and assembly
while ensu ing he p ope mechanical pe o mance o he sin e ed s uc u e in i o [49–51].
An example o he inal geome y o he pa ien -speci ic sca old is p o ided in he S1 File o
he Suppo ing In o ma ion. The p in ing nozzle diame e , he po e size, o he laye o e lap
we e also nume ically op imized in p e ious wo ks o maximize cell di usion and p oli e a-
ion while ensu ing he mechanical in eg i y o he s uc u e unde he o ine physiological
loads [17]. The inal mic oa chi ec u e had a po osi y o 59.3%, a 560.8 μm po e size, and a
speci ic su ace a ea o 5768.9 m
-1
[17]. A e d ying a oom empe a u e, he o ganic compo-
nen s o he implan we e elimina ed unde hea ing a 400˚C o 1 hou . They we e inally sin-
e ed a 1300˚C o 2 hou s o compac he pas e-like sca old. The chemical s e iliza ion o he
s uc u es was achie ed using o maldehyde a 60˚C and ela i e humidi y o 80%.
Fig 1. S eps in he design o he bone issue enginee ing su ge y. (A) Design o he bioce amic sca old om he 3D geome y o
he o ine me a a sus econs uc ed by compu ed omog aphy scans. (B) 3D-p in ing o he hyd oxyapa i e s uc u e using
oboscas ing. (C) Ha es ing o he spongy g a ing issue om he la e al side o he con ala e al hume us. (D) Implan a ion o he
sca old.
h ps://doi.o g/10.1371/jou nal.pone.0296510.g001
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Su gical p ocedu e
Be o e su ge y, Amoxiciline 15 mg/kg Clamoxyl
1
IM and Meloxicam 0.2 mg/kg Me acam
1
IV we e supplied o he animals o p e en in ec ions and excessi e in lamma ion. Su ge y was
ca ied ou unde gene al anes hesia induced by de omidine 20 μg/kg De ogesic
1
IV and mo -
phine 0.2 mg/kg Mo ina B.B aun
1
IM and was main ained wi h he inhala ional anes he ic
iso lu ane IsoVe 11–1.2% anspo ed in 100% oxygen. The sheep’s body empe a u e, blood
p essu e, oxygen sa u a ion, expi ed ac ion o ca bon dioxide and elec oca diog ams we e
cons an ly moni o ed du ing he in e en ion.
The animal was placed in igh la e al decubi us o a medial app oach. The igh hindlimb
was sha ed, and i s skin was asep ically p epa ed using chlo hexidine and an an isep ic alco-
hol-based solu ion. An 8-cm incision was hen pe o med using an elec ical scalp o expose,
a e a ca e ul sepa a ion o he pe ios eal issue, he unde lying me a a sal segmen o eplace.
Be o e os eo omizing, an Iliza o - ype ex e nal ixa o was implan ed in he me a a sus o
keep he esul ing unconnec ed bone agmen s aligned. This s abilizing ame comp ises wo
aluminum igs in e connec ed by me allic ba s and ixed o he bone h ough a o al o six
d illed 4-mm ØSchanz pins. Mo e in o ma ion abou he ex e nal ixa ion design and i s
mechanical p ope ies is p o ided in Bla
´zquez-Ca mona e al. [52]. Two pa allel ans e sal
os eo omies 15 mm apa om each o he we e made in he in e media e pa using an oscil-
la ing saw. As a esul , a c i ical-size 15 mm de ec was c ea ed in each animal. Adding bone
mo phogene ic p o eins o g a ed issue o he sca old is an ex ended s a egy used in issue
enginee ing expe imen s o a oid a lack o spon aneous healing in c i ical-size de ec s [21,22].
In his line, cancellous bone au og a om he la e al side o he con ala e al hume us head
was immedia ely ha es ed h ough a 7-cm incision using Volkmann spoons, as shown in Fig
1C. This spongy issue was inse ed in he inne hole o he sca old, wo king as a cell-seeding
ehicle o accele a e os eogenesis. The sca old was hen implan ed in o he de ec and ixed o
he dis al agmen by in oducing he couple in o he bone ma ow, as illus a ed in Fig 1D.
Since he sca old’s main body was 13 mm long, he emaining 2 mm p oximal gap was illed
wi h he emaining cancellous g a ing issue. A e a quick adiological analysis o e i y he
sca old implan a ion and he non-join in asion o he ixa o ’s pins, he ope a ed sheep we e
eco e ed wi h oxygen unde assis ance. Du ing he i s i e days a e su ge y, analgesia was
also p o ided (meloxicam 0.1 mg/kg SC Me acam
1
and bup eno phine 0.03 mg/kg IM
Bupaq1) acco ding o uminan pain scales. Du ing he esea ch, ex e nal ixa o s we e pe i-
odically checked, and he skin-pin ansi ions we e cleaned wi h chlo hexidine Desinclo 1.
Gai analysis
Gai es s consis ed o making he sheep walk o e a guided ci cula gangway con aining a
wi eless p essu e-sensi i e pla o m Pasco PS-2141
1
(PASCO, Rose ille, CA, USA), as can be
seen in Fig 2A. The load pla o m (35 x 35 cm) con inuously measu es he dynamic e ical
GRF in he animals’ s ance phases (sampling a e o 50 Hz), he la ges componen o he o al
GRF [53]. The pla o m was embedded inside he walking gai o isola e ib a ional noises and
enable measu ing ‘‘s eady-s a e” walking. Beyond he ope a ed igh ipsila e al hindlimb (IH),
limb gai condi ions we e also analyzed o assess po en ial compensa ion mechanisms o loss
o bea ing capaci y a e issue enginee ing in e en ion: he ipsila e al o elimb (IF), he con-
ala e al hindlimb (CH), and he con ala e al o elimb (CF). Fig 2B shows a scheme o hese
gai pa ame e s on a con ol GRF cu e. P io o su ge y, an acclima ion pe iod was also
needed o each animal since hey a e na u ally g ega ious animals, hus being a e se o walk-
ing in isola ed se ings [54]. A e su ge y and a la ency and eco e y pe iod o 7 days, weekly
measu emen sessions we e ca ied ou by eco ding 7–10 s ance phases pe limb, hus
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analyzing he da a as he daily a e age. Each gai phase o e e y limb was de e mined by ana-
lyzing he collec ed da a, s a ing om he ime-poin when an inc ease in g ound eac ion
o ce was eco ded un il he ime-poin a which i e u ned o 0 N. As inclusion c i e ia,
acqui ed s ance phases pe o med abo e o below amble speed (2–4 km/h ) we e isually dis-
ca ded. Mo eo e , da a in which he animal in e up ed i s ma ch a any poin o he gangway
we e no included in he analysis ei he . Du ing he expe imen al es s, a supe iso me icu-
lously w o e down he speci ic ime-poin s a which each limb execu ed a alid ull s ance
phase on he load pla o m o u he associa e each GRF cu e o hei espec i e limb. The
g ound eac ion o ce da a om hei eco ded s ance phases is p o ided in he S2 File o he
Suppo ing In o ma ion.
As shown in Fig 2B, ou main dynamic and kinema ic pa ame e s we e analyzed in e e y
collec ed s ance phase pe limb: he maximum g ound eac ion o ce (GRF
peak
), he a e age
g ound eac ion o ce in he s ance phase (GRF
mean
), he con ac ime wi h he g ound (
c
),
and he ela i e impulse (Imp) calcula ed as he a ea unde he GRF no malized by
c
o decou-
ple he e ec o gai speed. All he abo e pa ame e s a e s anda d in gai analysis expe imen s
wi h load pla o ms [24,38,39]. They we e addi ionally no malized by he body weigh o each
specimen (BW) o emo e body-size dependence. Consequen ly, he weigh o he animal was
con olled h oughou he en i e expe imen a ion pe iod, especially du ing he i s weeks
a e su ge y when an appe i e and a sligh weigh loss we e commonly su e ed by sheep. Fu -
he mo e, gai asymme y be ween ipsila e al and con ala e al limbs was assessed o each
p e ious gai pa ame e acco ding o he o mula ion employed in p e ious wo ks [39,55].
This index o he pa ame e xcan be calcula ed using Eq 1.
Asymme yð%Þ ¼ 100 �j XIXC
0:5�ðXIþXCÞj ð1Þ
whe e X
I
and X
C
a e he daily mean alues o each gai pa ame e o he ipsila e al and he
con ala e al limbs, espec i ely. The asymme y index should be close o ze o bo h o hind-
and o elimbs in heal hy specimens wi hou any o hopedic pa hology.
Fig 2. Gai es s and analyzed pa ame e s. (A) One sheep o he s udy walking in he ins umen ed gangway con aining he wi eless load
pla o m. (B) scheme o he measu ed gai pa ame e s o e a heal hy g ound eac ion o ce cu e (GRF) no malized by he bodyweigh o
he sheep.
h ps://doi.o g/10.1371/jou nal.pone.0296510.g002
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S a is ical analysis was also pe o med using he so wa e MATLAB
1
(The Ma hWo ks
Inc., Na ick, MA, USA) o assess he po en ial dependency be ween all analyzed gai pa ame-
e s o he ou limbs. P ecisely, he goodness-o - i o linea eg ession models was measu ed
h ough he coe icien o de e mina ion, R-squa ed and p- alue. The coe icien o de e mina-
ion R-squa ed is adi ionally in e p e ed as he pe cen age o a iance in one a iable p e-
dic ed o explained by he o he [56]. In his ield, au ho s end o in e p e R-squa ed o e
0.49 and 0.81 as a s ong and e y s ong co ela ion, espec i ely [5,38,39]. The p- alues o
he coe icien s indica e whe he hese p e ious ela ionships a e o no s a is ically signi ican .
Thus, a low p- alue (<0.01) would con i m ha one gai pa ame e is signi ican ly dependen
o ano he .
X- ay ollow-up
In pa allel wi h he gai analysis, he egene a ion p og ess was e i ied h ough mon hly x-
ays o he ope a ed me a a sal. These images we e aken o clinically ensu e he non-appea -
ance o non-unions and o quali a i ely associa e changes de ec ed in gai condi ions wi h he
le el o ossi ica ion a he di e en egene a ion s ages.
Resul s
Gai pa ame e s no malize o e healing ime
A e a week o pos -su gical eco e y and la ency, he sheep we e able o walk app op ia ely
on he ins umen ed ga e. In gene al, he weigh o he animals dec eased om 57.90 ±9.62 kg
a e su ge y o 53.04 ±6.34 kg be o e he sac i ice. Fig 3 compa es he e olu ion o he GRF,
no malized by he body weigh (% BW), du ing gai es s a di e en ime poin s o he consol-
ida ion phase, speci ically weeks 3, 10, and 30 a e su ge y. Fo one expe imen al es in hese
weeks o one o he specimens, he cu es show he means (do ed black lines) and s anda d
de ia ions o he o ce o he 7–8 s ance phases o all limbs: ipsila e al and con ala e al o e-
limbs (IF and CF, blue cu es) and hindlimbs (IH and CH, ed cu es). No e ha he ope a ed
me a a sal in which he bioce amic sca old eplaced a c i ical-size bone agmen was he ipsi-
la e al hindlimb (IH). The heal hy con ol g oup da a a e also included (g een cu es). Com-
pa ing wi h hese con ol cu es, a ema kable al e a ion in gai condi ions and limbs’ loading
capaci y o he animal was ound in he i s weeks a e su ge y. Fi s , he pe cen age o body
weigh ca ied by he ope a ed IH was educed by app oxima ely 10%. Fu he mo e, simila ly
o humans, he con ol e ical o ce da a o he sheep’s hind limbs du ing a gai cycle has a
e y pa icula M-shape wi h ou inc easing and dec easing in e als. This beha io is unda-
men ally due o ini ial mo emen s and accele a ions o he animals’ cen e o mass in he body
weigh dis ibu ion wi h he o elimbs, as well as he appea ance o a p opulsi e ac ion be o e
push-o . This M-shape comple ely disappea ed a e su ge y, epo ing a single maximum
GRF o punc ual con ac wi h he g ound. In pa allel, he es o he limbs ca ied sligh ly
mo e GRF du ing hei s ance phases a he beginning o he egene a ion p ocess (Fig 3, week
3): 11.2% o he BW abo e con ol in IF, 21.7% in CF, and 17.59% in CH. None heless, he
shape o he o ce cu es was no d as ically modi ied as he ope a ed limb’s. Fig 3 also shows
ha all limbs no malized he dis ibu ion o body weigh and he bea ing capaci y o hei
limbs o heal hy da a h oughou he consolida ion weeks. Fo ins ance, he ope a ed IH limb
epo ed alues simila o hose o he con ol g oup a week 30, wi h 40% BW compa ed o
44% in non-ope a ed sheep. Howe e , he GRF cu e o he IH limb did no egain he heal hy
M-shape in he medium e m. Simul aneous o his eco e y, he x- ays o Fig 3 show a g adual
ossi ica ion o he c i ical-size bone de ec and he g adual o ma ion o a issue b idge ha
consis en ly mine alized, connec ing he o iginal p oximal and dis al bone agmen s.
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Simila ends we e ound when compa ing he e olu ion o all he gai pa ame e s analyzed
o all he animals. The comple e se o expe imen al da a o hese pa ame e s is p o ided in
he S3 File o he Suppo ing In o ma ion. Fo example, Fig 4A and 4B analyze he e olu ion
o he GRF
peak
and GRF
mean
in he ou limbs: ope a ed IH limb ( ed emp y ma ke ), IF limb
(blue emp y ma ke ), CH limb ( ed illed ma ke ), and CF (blue illed ma ke ). The means o
he con ol da a o he o e- and hindlimbs a e also ep esen ed as do ed lines. Bo h pa ame-
e s signi ican ly educed he GRF le els a e he TE su ge y. Speci ically, he eplacemen o
he bone agmen by he bioce amic sca old educed he GRF
peak
o 15–23% o he BW (57–
163.46 N depending on he animal) and he GRF
mean
o 11–16% (39.52–95.13 N) when he
con ol alues we e a ound 42% and 28% BW, espec i ely. These pa ame e s ook a ound 160
days o e u n o heal hy alues. The con ala e al limb ollowed he in e se end s a ing
om 44–62% o he BW o he GRF
peak
pa ame e (242.97–329.67 N depending on he ani-
mal) and om 30–39% o he BW o he GRF
mean
(158.42–224.49 N). Likewise, he o elimbs
also ini ially ca ied mo e body weigh han hei co esponding con ol du ing gai , 65% and
44% o he BW (a ound 370 and 250 N) o GRF
peak
and GRF
mean
, espec i ely. As e lec ed in
Fig 4C, he egene a ion p ocess also impac ed he
c
. Al hough he s epping ime o a sheep is,
on a e age, 0.58 s in hindlimbs and 0.52 s in o elimbs, depending on he specimen, he in e -
ened limbs made con ac wi h he g ound be ween 0.25–0.47 s. In con as , o he limb’s
Fig 3. E olu ion o he g ound eac ion o ce cu es o e he weeks a e su ge y. G ound eac ion o ce cu es (GRF) o e
he s ance phases o he ipsila e al o elimb (IF), he ope a ed igh ipsila e al hindlimb (IH), he con ala e al o elimb (CF),
and he con ala e al hindlimb (CH). Weeks a e su ge y and x- ays a e included as a e e ence o he deg ee o ossi ica ion a
said ime-poin . Con ol g ound eac ion o ce da a is included o all limbs (g een cu es).
h ps://doi.o g/10.1371/jou nal.pone.0296510.g003
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